Despite aggressive management, septic shock arising from Gram-negative bacteria sepsis continues to be a leading cause of death in both surgical and medical patients. Death in such patients usually results from cardiovascular collapse and/or multiple organ system failure. One of the most numerous and dominant agents causing sepsis from Gram-negative bacterial infection is endotoxin, which is present on the surface of Gram-negative bacteria, including Escherichia coli. 
Bacterial endotoxin is a complex consisting of lipid, carbohydrate and protein. It is characterized by an overall negative charge, heat stability and high molecular weight. Highly purified endotoxin does not contain protein, and is comprised of lipopolysaccharide (LPS) and lipooligosaccharide (LOS). Depyrogenation can generally be achieved by inactivating or removing endotoxin, depending upon the physicochemical nature of the LPS. LPS consists of three distinct chemical regions, lipid A, which is the innermost region, an intermediate core polysaccharide, and an outermost O-specific polysaccharide side chain, which is responsible for an endotoxin's particular immunospecificity.
Bacterial endotoxins present severe pathophysiological reactions when introduced into animals, including high fever, vasodilation, diarrhea and, in extreme cases, fatal shock. (Morrison, 1987). Thus, it is critical to avoid endotoxin contamination in any pharmaceutical product or medical device which comes into contact with body fluids. In addition, high endotoxin levels in sera due to bacterial diseases, such as septicemia, are not easily treated. Antibiotic treatment of the infection only kills the bacteria, leaving the endotoxin from their cell walls free to cause fever.
Endotoxins tend to form micellar structures which have a similar density, size, and charge distribution on the outer surface of the micelles. As a result, endotoxins co-purify with proteins or nucleic acids. Various attempts have been made to eliminate endotoxins present in biological and pharmaceutical compositions (U.S. Pat. Nos. 5,972,225; 6,132,610; 6,194,562; 5,747,455; 5,169,535; 5,101,019; 4,808,314; 4,059,512 and 3,959,128) and it has become increasingly evident, that endotoxins are not readily separated from protein or nucleic acid samples. Further, endotoxins are extremely stable, resist extremes of temperature and pH value and have a broad spectrum of biological activity (e.g., are toxic in humans and other animals, are pyrogenic when present in trace amounts, and can cause hypotensive shock, disseminated intravascular coagulation and death).
Bacterial endotoxins thus impede progress in various areas of biotechnology. Gram-negative bacteria can shed endotoxins from their cell walls and endotoxins are therefore a potential contaminant of any aqueous solution. For example, during lysis of bacterial cells, such as is done in recombinant protein purification or to release plasmids from transformants (e.g., E. coli), endotoxins are released into the lysate produced thereby. Endotoxin contamination in protein or nucleic acid samples can adversely limit the utility of the sample, particularly in applications which are sensitive to such contamination (e.g., pharmaceutical compositions). For example, the transfection efficiencies of several different cultured eukaryotic cell lines, including HeLa, Huh7, COS7, and LMH, have been shown to be sharply reduced in the presence of endotoxins (Weber et al., 1995). Endotoxins have also been found to be toxic to primary human cells, such as primary human skin fibroblasts and primary human melanoma cells, in the presence of entry-competent adenovirus particles (Cotton et al., 1994).
Although glassware, plasticware, water, and most buffers can be effectively decontaminated from free endotoxins (see for example, Sofer, 1984; Issekutz, 1983), many proteinaceous macromolecules such as hormones, immunoglobulins, and enzymes are biologically inactive following such treatments. This is a particularly important problem with the recent advances in biotechnology. Bacterial contamination of useful biological products is recognized as a problem (Wightsmith et al., 1982). Endotoxin-producing bacteria used in genetic engineering experiments can add greatly to the risk of endotoxin contamination of materials produced by such techniques.
Ultrafiltration, dialysis and certain chromatographic methods have been employed to remove endotoxin from aqueous solutions. These methods typically separate small molecules from endotoxins based on the size difference between the small molecule and endotoxin, which aggregates into high molecular weight micelles in aqueous solutions. However, endotoxins and many macromolecules are often too similar in size to be separated using these techniques. Additional chromatographic purification techniques, such as adsorbing matrices, affinity chromatography and ion exchange chromatography, have been described to remove endotoxin in a solution (U.S. Pat. Nos. 3,897,309; 4,276,050; 4,381,239; Morrison et al., 1976; Duff et al., 1982; Issekutz, 1983). However, these procedures typically still permit about 10% of the originally present endotoxin to remain in solution or associated with protein following elution from the column (e.g., see Duff et al., 1982). The presence of that 10% protein-associated endotoxin may not affect the endotoxin assay, but still could remain pyrogenic.
Moraxella catarrhalis is an important human respiratory tract pathogen. M. catarrhalis is the third most common cause of otitis media in infants and children (Murphy, 1989). Moraxella catarrhalis is a common cause of sinusitis and conjunctivitis in both children and adults (see e.g., Bluestone, 1986; Brorson et al., 1976; Romberger et al., 1987) and is an important cause of lower respiratory tract infections in adults with chronic bronchitis and chronic obstructive pulmonary disease (Murphy et al., 1992; Catlin, 1990). Additionally, M. catarrhalis can cause pneumonia, endocarditis, septicemia, and meningitis in immunocomprised hosts (Cocchi et al., 1968; Douer et al., 1977; McNeely et al., 1976).
Since recurrent otitis media is associated with substantial morbidity, and the attendant health care costs, there is interest in developing strategies for identifying and preventing these infections. One such approach is the development of immunogenic compositions for preventing bacterial otitis media. Outer membrane proteins are being investigated as antigens having utility in diagnosing and immunizing against disease caused by bacterial pathogens, such as M. catarrhalis. However, it is imperative in the formulation of these compositions, that the outer membrane protein antigen(s) is effectively free of bacterial endotoxin, so as to prevent sepsis.
Thus, there is presently a need for simple and efficient methods or processes to purify protein samples, effectively free of bacterial endotoxin. It is additionally desirable that such a protein purification method occurs without significant loss in protein concentration or biological activity, such that the protein can be administered (e.g., parenterally) as a pharmaceutical or immunogenic composition, effectively free of endotoxin.